Orbitrap Collision Cross Section Measurements Enhance Isomer Annotations in Lipidomics

Despite advances in high-resolution mass spectrometry (HRMS), confident lipid annotation remains challenging due to the extensive chemical diversity of the lipidome and the prevalence of isomeric species. Ion mobility collision cross section (CCS) measurements provide structural information that complements HRMS; however, not all HRMS platforms can perform these measurements, necessitating a trade-off among mass resolution, accuracy, and robustness. Here, we introduce a method to infer lipid CCS values directly from liquid chromatography (LC)-Orbitrap MS experiments (OrbiCCS). We show that Orbitrap mass analyzer pressure readings, and therefore CCS values, are influenced by the LC gradient solvent composition, requiring correction using isotopically labeled internal standards injected post-column. We also show that hundreds of lipid features can be assigned OrbiCCS values in a single LC run, with average precision better than 1% and an accuracy of 1-2% relative to reference DTCCS and TIMSCCS values. This excellent CCS accuracy not only enables more reliable annotation of lipid species in complex mixtures by matching OrbiCCS values to reference databases but also accelerates lipid structural elucidation based on the unknowns position in OrbiCCS-retention time-m/z space.